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Journal of Bacteriology, January 1999, p. 389-395, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rhizobium (Sinorhizobium)
meliloti phn Genes: Characterization and Identification of
Their Protein Products
George F.
Parker,1
Timothy P.
Higgins,1
Timothy
Hawkes,2 and
Robert L.
Robson1,*
School of Animal and Microbial Sciences,
University of Reading, Reading RG6 6AJ,1 and
Zeneca Agrochemicals, BioScience, Bracknell RG42
6ET,2 United Kingdom
Received 3 August 1998/Accepted 25 September 1998
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ABSTRACT |
In Escherichia coli, the phn operon encodes
proteins responsible for the uptake and breakdown of phosphonates. The
C-P (carbon-phosphorus) lyase enzyme encoded by this operon which
catalyzes the cleavage of C-P bonds in phosphonates has been
recalcitrant to biochemical characterization. To advance the
understanding of this enzyme, we have cloned DNA from
Rhizobium (Sinorhizobium) meliloti
that contains homologues of the E. coli phnG,
-H, -I, -J, and -K
genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P
lyase. Furthermore, the phenotype of this phn mutant shows
that the C-P lyase has a broad substrate specificity and that the
organism has another enzyme that degrades aminoethylphosphonate. A
comparison of the R. meliloti and E. coli phn
genes and their predicted products gave new information about C-P
lyase. The putative R. meliloti PhnG, PhnH, and PhnK
proteins were overexpressed and used to make polyclonal antibodies.
Proteins of the correct molecular weight that react with these
antibodies are expressed by R. meliloti grown with
phosphonates as sole phosphorus sources. This is the first in vivo
demonstration of the existence of these hitherto hypothetical Phn proteins.
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INTRODUCTION |
Phosphonates are organophosphorus
compounds containing the chemically inert carbon-phosphorus (C-P) bond.
Examples of naturally occurring phosphonates include
phosphoenolpyruvate, 2-aminoethylphosphonate (2-AEP), and
phosphonoacetate (PA) (16). In addition to these natural
compounds, man-made phosphonates are now entering the environment in
significant quantities (7). The ability to degrade phosphonates is relatively widespread, occurring in gram-positive (22, 39) and gram-negative bacteria (8, 39) as
well as in fungi (20). Three classes of enzyme capable of
breaking the C-P bond of phosphonates are known: PA hydrolase, an
enzyme specific for PA breakdown (27, 30); phosphonatase,
which specifically degrades 2-AEP (22, 24); and C-P lyase,
which cleaves the C-P bond in a broad spectrum of phosphonates
(10). C-P lyase activity can be detected in whole organisms;
however, it has never been convincingly assayed in cell extracts
(43), and this has limited attempts to understand the
mechanism of the enzyme, which has been suggested to involve a
redox-dependent free radical mechanism (10). The uptake and
breakdown of phosphonates in Escherichia coli is, however,
well characterized genetically (4). The phn gene
cluster consists of 17 genes (phnA to -Q), of
which phnC to -P appear to be required for
phosphonate uptake and breakdown (33). Mutagenesis of the
phn gene cluster revealed that phnCDE encode a
phosphonate transporter, phnF and phnO may have
regulatory functions, phnG to -M are likely to be
components of the C-P lyase, and phnN and phnP
are probably accessory proteins (34). To broaden knowledge
of C-P lyase, we chose to work with Rhizobium
(Sinorhizobium [6]) meliloti
because (i) it contains a C-P lyase able to degrade the important
herbicide N-phosphonomethyl-glycine (25), (ii) some phn genes in this organism have been sequenced
(28), and (iii) a phosphate/phosphonate transporter, encoded
by phoCDET, is needed for the successful formation of a
symbiotic association between this bacterium and alfalfa
(Medicago sativa) (1).
In this study, we cloned DNA encoding the putative phn genes
from R. meliloti, constructed a mutation in the genome which establishes a role for these genes in phosphonate degradation, and
demonstrated for the first time that proteins deduced to be encoded by
phn genes are produced in vivo by organisms growing with
phosphonates as the sole phosphorus sources.
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MATERIALS AND METHODS |
Sources of reagents.
Methylphosphonate (98%),
ethylphosphonate (97%), propylphosphonate (95%),
tert-butylphosphonate (98%), aminomethylphosphonate (99%),
3-aminopropylphosphonate, glyphosate (phosphonomethylglycine) (96%),
phenylphosphonate (98%), and 4-aminobenzylphosphonate (97%) were
obtained from Aldrich Chemical Company (Dorset, England). 2-AEP (99%),
glycerol-3-phosphate (95%), and PA (98%) came from Sigma-Aldrich Ltd.
(Dorset, England). Benzylphosphonate (97%) was from Lancaster
Synthesis Ltd. (Lancashire, England).
Bacterial strains and growth conditions.
Strains used in
this work are described in Table 1.
R. meliloti was grown at 30°C either on TY (3)
with 6 mM CaCl2 or on acid minimal salts (36)
modified by increasing the CaCl2 concentration to 1.2 mM
and adding nicotinic acid (1 mg/liter); the carbon source was 50 mM
succinate, and phosphorus sources were provided at 0.5 mM unless
otherwise stated. To make solid acid minimal salts medium containing a
low level of inorganic phosphate, the medium was made double strength
and added to a molten solution of 1.8% (wt/vol) agarose. E. coli was grown on LB (35). Antibiotics for R. meliloti were added at 500 (streptomycin), 20 (spectinomycin), and
10 (gentamicin) µg/ml; those for E. coli: were added at
100 (ampicillin), 20 (kanamycin), and 5 (gentamicin) µg/ml.
Genetic and DNA manipulations and sequencing.
All routine
DNA manipulation and analysis were done as described in reference
40. Southern transfer to Amersham Hybond N nylon
membrane and detection of hybrids by using the Boehringer Mannheim
digoxigenin chemiluminescence kit were performed according to the
manufacturer's instructions. Probes were made by PCR amplification of
plasmid inserts with digoxigenin-labeled dUTP in the mixture. Genetic
conjugations were done by triparental matings according to the method
of Figurski and Helinski (9), with E. coli DH5
as the donor strain and DH5(pRK2013) used for the transfer
functions. Transconjugants were selected on TY agar containing
streptomycin and spectinomycin.
Nucleotide sequences were obtained by automated sequencing using a
Pharmacia ALF express DNA sequencer. The sequencing reactions were done
with an Amersham Thermosequenase kit according to the manufacturer's
instructions with Cy5-labeled primers.
PCR.
Oligonucleotide primers are described in Table
2. Reaction mixtures contained primers
(0.2 µM), deoxynucleoside triphosphates (0.25 µM),
MgCl2 (2 mM), target DNA (0.5 µg), dimethyl sulfoxide (10%, vol/vol) 1× OptiPerform buffer (Bioline UK Ltd.), and 2 U of
Bio-X-Act DNA polymerase (Bioline UK Ltd.) in a final volume of 50 µl. The mixture was overlaid with mineral oil and subjected to 30 cycles of 95°C for 30 s, 60°C for 30 s, and 68°C for 1 min/expected kb of product.
Sequence analysis.
Use was made of the Genetics Computer
Group (GCG) Wisconsin Package, release 8.1 (11). DNA
sequences were assembled by using the GelAssemble package. Scans for
expressed genes were done with TestCode and CodonFrequency, using an
R. meliloti codon preference table based on GenBank release
104 from the codon usage database at the National Institute of
Agrobiological Resources, Tsukuba, Japan (http://www.dna.affrc.go.jp/).
Homology searches were done with the GCG implementation of FASTA and
with BLAST (12) implemented at the National Biotechnology
Information Center, using the default settings of both algorithms.
Scans for sequence motifs were done with the scan ProSite tool at the
Swiss Institute of Bioinformatics (http: //www.expasy.ch/) and
GeneFind (44)
(http://athena.uthct.edu/cgi-bin/genefind.pl). Multiple sequence
alignments were done with Pileup (GCG), Multalin (5), and
ClustalW (14).
Cloning and overexpression of putative phn genes of
R. meliloti.
PCR primers (Table 2) were designed to amplify
the nucleotide sequences of the genes encoding the putative Phn
proteins. Restriction sites were added at the 5' end of each primer for cloning purposes. Individual genes were amplified by PCR, and the
resultant products were cloned into the pRSETB expression vector (Table
1). Two expression constructs were made for each putative gene. First,
translational fusions between the N-terminal coding sequence of each
gene product and the polyhistidine tag coding sequence of pRSETB were
constructed. The second set of constructs fused the ATG start codon of
the putative phn genes and the NdeI site of the
pRSETB vector, giving plasmids potentially capable of expressing native
gene products. However, the putative phnJ gene contained an
internal NdeI site, which was removed by PCR as follows. A
5' fragment of the gene was amplified by using primers PhnJF and
PhnJiR, which contains an A-to-G substitution that removes the
NdeI site without altering the predicted protein product. An
overlapping 3' fragment was amplified by using the primers PhnJiF and
PhnJR (Table 2; Fig. 1). The two PCR
products were ligated after digestion with EarI, which cuts
at a unique restriction site in the gene. The ligated product was then
cloned into pRSETB as described above. Both sets of constructs were
electroporated into E. coli DH5 and E. coli
BL21(DE3). The E. coli BL21(DE3) clones were
subsequently used in overexpression experiments. Cells were grown
overnight at 37°C with shaking in LB medium (10 ml) supplemented with
ampicillin (100 µg/ml). Overnight cultures were used to inoculate
fresh media, which were grown at 37°C to an optical density at 600 nm
of 0.4 to 0.7. Isopropyl-
-D-thiogalactopyranoside (IPTG;
1 mM, final concentration) was added to the cultures, which were grown
for a further 2 h prior to harvesting.
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FIG. 1.
Inferred physical map of the DNA sequence of the
R. meliloti phn gene cluster (28) showing
locations of the PCR primers used in this study (small arrows above the
sequence) and the predicted protein products. The 2-kb
Spr-Smr fragment from plasmid pHP45
(37) was inserted into the DNA cloned in pRMP1 as a
SmaI fragment into the ClaI and NdeI
sites blunted with the Klenow fragment of DNA polymerase I
(40). This construct was inserted into the XbaI
site of pJQ200SK to give pRMP4, which was conjugated into R. meliloti to gave the mutant strain Pn1.
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Analytical methods.
Cell extracts were prepared by
harvesting early- to mid-log-phase cells of R. meliloti
grown on various phosphorus sources. Cells were washed twice in 50 mM
Tris-HCl buffer (pH 7.5), resuspended in the same buffer to 1/10 the
original culture volume, and sonicated on ice. Cell debris was removed
by centrifugation at 3,000 × g for 15 min at 4°C,
and the extracts were stored at 
20°C until required. The protein
content of cell extracts was estimated by the method of Lowry et al.
(26), using bovine serum albumin as a standard. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
(21) was carried out on 12% (wt/vol) polyacrylamide gels.
Purification of His-tagged phn gene products was carried out
by affinity purification using a 1-ml HiTrap chelating column (Pharmacia Biotech). Extracts from postinduced E. coli
BL21(DE3) cells were prepared by harvesting and resuspension to
one-fifth the original culture volume in guanidinium lysis buffer (20 mM sodium phosphate, 0.5 M sodium chloride, 6 M guanidine hydrochloride [pH 7.8]), rocking for 5 to 10 min at room temperature, and
sonication on ice. Cell debris was removed by centrifugation at
3,000 × g for 15 min at 4°C, and extracts were
stored at 20°C until required. HiTrap columns were prepared by
washing with 5 column volumes (CV) of distilled water to remove the
20% ethanol storage buffer. The columns were primed by addition of 1 ml of 0.1M NiCl2 · 6H2O and then
equilibrated with 5 CV of denaturing binding buffer (20 mM sodium
phosphate, 0.5 M sodium chloride, 8 M urea [pH 7.8]); after loading
of cell extracts, the columns were washed with a further 5 CV of
denaturing binding buffer. The columns were then washed with 5 CV of
denaturing wash buffer (20 mM sodium phosphate, 0.5 M sodium chloride,
8 M urea [pH 5.3]) to remove contaminating nonrecombinant proteins.
Recombinant proteins were eluted from the columns with 5 CV of
denaturing elution buffer (20 mM sodium phosphate, 0.5 M sodium
chloride, 8 M urea [pH 4.0]). The purified proteins were then used to
raise polyclonal antibodies in rabbits.
Western immunoblotting was carried out essentially as described in
reference
40 except that blotted nitrocellulose
filters
were blocked overnight with blocking solution (50 mM Tris-HCl,
150 mM sodium chloride, 0.2% [vol/vol] Tween 20, and 5% [wt/vol]
nonfat dried milk powder) prior to addition of primary antibody
in the
same blocking solution. Primary antibodies were detected
with alkaline
phosphatase-conjugated goat anti-rabbit immunoglobulin
G (whole
molecule; Sigma) treated with CDP-star chemiluminescent
substrate
(Amersham International) as directed by
manufacturer.
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RESULTS |
Cloning of the R. meliloti phn genes.
The sequence
of a putative R. meliloti phn gene cluster (28)
was used to design PCR primers to amplify this region. Primers RmPhn1
and RmPhn2 (Fig. 1; Table 2) used in a PCR with R. meliloti genomic DNA gave an expected 3.9-kb product. Products from independent reactions were cloned to give plasmids pRMP1 and pRMP2, respectively. The inserts were restriction mapped to provide a comparison with the
submitted sequence (Fig. 1). We further confirmed by Southern hybridization that the cloned DNA represents the genome correctly (data
not shown). The DNA cloned into pRMP1 and pRMP2 was sequenced and found
to be identical to that already submitted (28) between nucleotides (nt) 268 and 4148, except for two differences (T to C at nt
852 and G to A at nt 926). TestCode prediction and comparison with a
codon usage table confirm that homologues of E. coli PhnG, PhnH, PhnI, PhnJ, and PhnK proteins are the most likely products encoded by this DNA (Table 3). However,
we predict that the PhnG protein is 34 amino acid residues longer at
the N terminus than that reported in GenBank (28). This
additional sequence shows good homology to the E. coli PhnG
protein.
PhnI and PhnJ are especially well conserved between
E. coli
and
R. meliloti (Table
3). It has been suggested that C-P
bond
cleavage by C-P lyase involves free radical- and redox-dependent
chemistry, possibly involving transition metals (
10). Metal
ions could therefore be of considerable importance in C-P lyase,
and
therefore it is interesting that alignments of PhnI reveal
that the
C-terminal domain contains histidinyl residues which
show an
arrangement resembling that of a lipoxygenase (Fig.
2a)
and which may provide ligands to
metal ions. Also, alignments
of PhnJ reveal four conserved cysteinyl
residues, which could
be ligands to a metal or metal sulfur cluster
site (Fig.
2b).
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FIG. 2.
(a) Alignment of E. coli PhnI (EcPhnI) and
R. meliloti PhnI (RmPhnI) proteins. The highlighted amino
acids are the additional C-terminal sequence on the R. meliloti protein and a region that shows homology to part of
lipoxygenase (LOX) L-2 of Oryza sativa (SwissProt accession
no. P29250) which contains the iron-binding region signature 2 (Prosite
PS00081). (b) Alignment of E. coli and R. meliloti PhnJ proteins. The four conserved cysteines are arrowed,
and the additional C-terminal sequence on the R. meliloti
protein is highlighted.
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Construction and characterization of a mutant in the putative
phn operon.
To discover if the DNA cloned as described
above is necessary for phosphonate utilization, we constructed a strain
which has a 2-kb deletion within this region of the genome (Fig. 1).
The presence of a genomic lesion was confirmed by Southern
hybridization and by PCR amplification of genomic DNA from the mutant
(data not shown). The phenotype of this mutation was assayed by
comparing the rates of growth in liquid minimal medium of the mutant
and wild type on several phosphonates and other compounds as sole sources of phosphorus (Table 4). This
mutation renders R. meliloti unable to utilize
aminomethylphosphonate, 3-aminopropylphosphonate, glyphosate, methylphosphonate, ethylphosphonate, propylphosphonate, phenylphosphonate, benzylphosphonate, 4-aminobenzylphosphonate, and PA.
The mutant strain is, however, still able to use 2-AEP and
glycerol-3-phosphate. This result demonstrates that the genomic lesion
it carries specifically affects phosphonate metabolism but does not
disrupt the PhoB-mediated phosphorus starvation response in this
organism (2, 29).
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TABLE 4.
Abilities of Rm1021 and Pn1 R. meliloti
strains to grow on a various phosphorus sources compared with results
of previous studies with E. coli
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Overexpression of the putative phn genes of R. meliloti.
We made expression constructs designed to produce
His-tagged and native forms of the putative Phn proteins (Table 1) and confirmed each structure by nucleotide sequencing (data not shown). Three of the five gene constructs, phnG, phnH,
and phnK, produced large amounts of recombinant protein
irrespective of the presence of the His tag (Fig.
3). Overexpression of putative PhnG, -H, and -K polypeptides lacking the N-terminal fusion resulted in the
production of recombinant proteins of 16, 22, and 26.9 kDa, respectively, in agreement with predicted values (Table 3). By contrast, no visible overexpression was achieved with the other two
gene constructs (phnI and phnJ [data not
shown]). Many reasons exist for the lack of expression of recombinant
proteins (41); however, the lack of expression of
phnI and phnJ of R. meliloti may be
significant. Possibly the whole C-P lyase complex is required to
stabilize these components of the complex. Polyclonal antibodies were
raised against the affinity-purified His-tagged recombinant proteins and used in subsequent experiments.
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FIG. 3.
Expression of phn genes in E. coli
BL21(DE3). Proteins were analyzed by SDS-PAGE as described in Materials
and Methods. Lanes contain cell extracts of E. coli
BL21(DE3) containing expression constructs induced with 1 mM IPTG.
Lanes: 1, pTHGB (PhnG with His tag); 2, pTHGC (PhnG without His tag);
3, pTHHB (PhnH with His tag); 4, pTHHC (PhnH without His tag); 5, pTHKB
(PhnK with His Tag); 6, pTHKC (PhnK without His tag). Molecular masses
(in kilodaltons) are shown to the left; expressed proteins are
indicated by arrows.
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Expression of putative phn genes in R. meliloti.
Western immunoblots were made of R. meliloti
cell extracts grown on liquid minimal media in the presence of various
phosphorus sources. The results show that the antibodies to the three
putative phn genes, phnG, -H and
-K, do indeed detect polypeptides of the predicted size
(Fig. 4). This finding suggests
that the genes do not undergo any major posttranslational
modification, e.g., removal of signal sequence. All three polyclonal
antisera gave consistent results in that bands corresponding to those
of the putative phn gene products were visualized only in
cell extracts of R. meliloti grown in the presence of
methanephosphonate, aminomethylphosphonate, and glyphosate as sole
sources of phosphorus. Bands were absent from cell extracts of R. meliloti grown in the presence or absence of inorganic phosphate
and aminoethylphosphonate. These data obtained from the Western blots
are consistent with those for R. meliloti Pn1 (Table 4) with
regard to growth and expression.
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FIG. 4.
Immunoblot analysis of cell extracts from R. meliloti grown on various phosphorus sources. Lanes: 1, no
phosphorus source; 2, inorganic phosphate; 3, methylphosphonate; 4, aminomethylphosphonate; 5, aminoethylphosphonate; 6, glyphosate. Lanes
7 to 9 contain cell extracts of E. coli BL21(DE3) containing
pTHGC (phnG), pTHHC (phnH), and pTHKC
(phnK) induced with 1 mM IPTG. Immunoblotting was done with
antisera raised against PhnG (a), antisera raised against PhnH (b), and
antisera raised against PhnK (c).
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Extracts from strain Pn1, induced by phosphorus starvation in the
presence of phosphonates, were also probed with the three
polyclonal
antisera. The mutant produces wild-type levels of PhnG,
reduced levels
of PhnH, and no detectable PhnK (data not shown).
This finding
demonstrates (i) that the mutation carried by Pn1
is polar, abolishing
expression of genes 3' to the insertion,
which suggests that these
genes are regulated by single promoter
as predicted in the original
sequence submission (
28), and (ii)
that PhnG is stable in
the absence of at least
phnI,
phnJ, and
phnK gene
products.
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DISCUSSION |
In this study, we confirm that the genome of R. meliloti contains a locus homologous to part of the phn
gene cluster of E. coli and that mutagenesis of this locus
prevents R. meliloti from degrading a wide variety of,
though not all, phosphonates. Antibodies raised to the putative PhnG,
PhnH, and PhnK proteins from R. meliloti were used to show
for the first time in any organism that these proteins are indeed
expressed. Moreover, their expression in R. meliloti was
seen only during growth with particular phosphonates as sole phosphorus
sources. These observations are consistent with R. meliloti
having a C-P lyase-type enzyme encoded by a phn gene cluster
similar, in part at least, to that of E. coli. This view is
substantiated by our observation that R. meliloti produces methane when growing with methylphosphonate as the sole phosphorus source (15) since the direct evolution of hydrocarbon
products from phosphonates is a hallmark of C-P lyase activity
(10, 42).
The C-P lyase enzyme of R. meliloti appears to have a
broader substrate specificity than its E. coli counterpart
because while R. meliloti can grow with benzylphosphonate
and glyphosate as sole phosphorus sources, E. coli cannot.
In the case of glyphosate, R. meliloti gives products
consistent with degradation involving C-P lyase activity
(25). Growth on a similar wide range of phosphonates has
been observed in Agrobacterium radiobacter (42)
and in Arthrobacter sp. strain GLP1 (18), where
the broad specificity was proposed to result from the activity of two
different C-P lyases. Our data suggest that R. meliloti has
a single C-P lyase which is able to degrade a wide range of phosphonates.
This work also sheds light on the variety of inducible
phosphonate-degrading enzymes that R. meliloti may express
under phosphorus starvation. R. meliloti, like
Enterobacter aerogenes (23), appears to have an
enzyme or enzymes other than C-P lyase that degrades 2-AEP, namely,
phosphonatase, because disruption of the phn gene cluster
does not prevent growth on 2-AEP. Also, phnG, -H,
and -K gene products were not expressed in R. meliloti 1021 growing with 2-AEP as a phosphorus source.
Therefore, the enzyme(s) that degrades 2-AEP appears to be
biochemically and genetically distinct from C-P lyase.
As regards the third class of C-P bond-cleaving enzyme known, PA
hydrolase (27), we have shown that R. meliloti
cannot induce this type of enzyme when using PA as its sole phosphorus
source because even the relatively slow growth with PA is abolished
when C-P lyase is inactivated by mutation.
A full understanding of C-P lyase function requires a better knowledge
of the role of each of the phn gene products. However, we
are unable to make any inferences about the function of individual genes from the phn mutation that we have constructed. Apart
from the deletion that we have shown, the mutation carried by strain Pn1 is polar. The absence of a candidate terminator downstream of the
R. meliloti phnK gene suggests the existence of more
phn genes downstream, and preliminary sequencing indicates
the presence of a gene homologous to E. coli phnL 3' to
phnK (15). Nonetheless, the availability of
antibodies specific to several putative phn gene products is
an important step toward further characterization of the C-P lyase
enzyme. The data presented in this report provide the first evidence
that proteins predicted to be part of the C-P lyase complex are
expressed in organisms provided with phosphonates as sole phosphorus
sources. These antibodies are useful tools for examining the hypotheses
about the properties, localization, and activity of C-P lyase.
| |
ACKNOWLEDGMENTS |
This work was funded by a grant from Zeneca Agrochemicals to
R.L.R.
All DNA sequencing was done by the Core Sequencing Facility, School of
Animal and Microbial Sciences, University of Reading. Polyclonal
antisera were raised by Andrew Dinsmore, Zeneca Pharmaceuticals, Cheshire, United Kingdom. We are heavily indebted to Philip Poole and
David Allaway (AMS, Reading, United Kingdom) for advice on how to work
with rhizobia and for supplying plasmids and to Frank Cannon for
communication of unpublished data. We are also grateful to Stephen
Cairns and Andrew Tingey for help and advice.
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FOOTNOTES |
*
Corresponding author. Mailing address: School of Animal
and Microbial Sciences, University of Reading, Whiteknights, P.O. Box
228, Reading RG6 6AJ, United Kingdom. Phone: (44) 1189 316639. Fax:
(44) 1189 316562. E-mail: sksrobsn{at}reading.ac.uk.
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Abstract
Introduction
